Global transcriptome analysis of THP-1 cells infected with an rpfB gene knockout strain of Mycobacterium tuberculosis.

Masters Degree. University of KwaZulu-Natal, Durban.Background: Since the initial isolation of Mycobacterium tuberculosis (Mtb), tuberculosis (TB) remains among the top 10 causes of death. In 2019, 10 million people globally were burdened with TB, of which 25 %were from Africa. A fundamental process in determining the outcome of Mtb infection is host-pathogen interactions. These interactions may lead to eradication of bacteria via the innate immune response or latent TB infection (LTBI) in which the bacilli reside in a non-replicating, low metabolic state (dormancy) within alveolar macrophages (AM). Ten percent of the global population develops LTBI leaving them at greater risk of developing active TB. Initiation of an immune response requires effective antigenic stimulation that induces multiple signalling cascades and the production of pro-inflammatory and anti-inflammatory cytokines, effective against or beneficial to Mtb. The pathogen has a family of 5 resuscitation promoting factors (rfps A-E) that have been previously characterized in Mtb growth, persistence and reactivation, in vitro and in vivo, using single and multiple rpf gene knockouts. Among the other Rpf proteins, RpfB was shown to be highly antigenic and immunogenic, with the ability to induce a Th1 phenotype immune response in dendritic cells (DC) through binding of toll-like receptor 4 (TLR4). Since macrophages are the target cell of Mtb and the reservoir of dormant bacilli, this study investigated the effect of rpfB in THP-1 macrophages at a late phase of replication (72 hours post-infection) using an Mtb rpfB gene knockout strain, through global transcriptomic analysis. Material and Methods: THP-1 macrophages seeded at 7.5 x 105 cells/ mL, were infected at a MOI of 1 with wildtype (WT), ΔrpfB mutant and rpfB-complemented strains. To elucidate host transcriptomic changes attributed to rpfB, mammalian RNA was isolated 72 hours post-infection (P.I) and sequenced using the Illumina High Seq platform. Bioinformatics analysis was performed using the tuxedo suite and Ingenuity Pathway Analysis (IPA). THP-1 cytokine production was analysed at 24-, 48-, and 72 hours P.I using the human GM-CSF and IL-1β multiplex panel (Bio-Rad Laboratories) in a Bio-Plex 200 System (Bio-Rad Laboratories). RNA sequencing data was validated using quantitative real-time PCR (qRT-PCR). Results: Global transcriptomic analysis revealed that rpfB induced differential transcriptional regulation in THP-1 macrophages. The total number of significantly enriched genes (SDEGs) induced by the WT strain was 5 times more than the mutant. Furthermore, the WT also induced a greater number of networks and upstream regulators. In addition this study showed rpfB stimulates, but is not essential to, IFN signalling, Role of JAK family kinases in IL-6 type cytokine signalling and Activation of IRF by Cytosolic PRRs, enhances IL-10 signalling and DC maturation, and enriches Acute phase response signalling, Phagosome formation, LXR/RXR activation, CAMP-mediated signalling, Gαi signalling and GADD45 signalling. In summary, the findings suggest rpfB contributes to the host protective immune response against Mtb infection. Upstream regulators induced by both infection models were all cytokines, however, they regulated genes that were exclusive to each pathway and mostly interferon stimulated genes (ISGs) in WT infected macrophages. Discussion and conclusion: Transcriptomic analysis showed rpfB induced recognition of host immunity and enhanced the THP-1 macrophage host immune response during late stages of Mtb infection. These findings provide basic evidence, which requires further interrogation, that rpfB regulates the immune response through interferon signalling and possibly plays a role in IL-10 signalling and DC maturation. Collectively, these findings indicate rpfB contributes to protective immunity of THP-1 macrophages against Mtb and may be used as a recombinant in vaccines against active TB, and in combination with LTBI antigens, against LTBI. However, since these observations are reported for the first time, further investigation to validate these findings would be valuable.Author's Dedication on page ii